The Rho activator ECT2 functions as a key regulator in cytokinesis. ECT2 is phosphorylated during G2/M phase, but the physiological significance of this event is not well known. In this period, we examined ECT2 phosphorylation in detail.First, we showed that phosphorylation of ECT2 at threonine-341 (T341) affects the auto-regulatory mechanism of ECT2. In G2/M phase, ECT2 was phosphorylated at T341 most likely by Cyclin B/Cyclin-dependent kinase 1 (Cdk1), and then dephosphorylated before cytokinesis. Depletion of ECT2 by RNA interference efficiently induced multinucleate cells. Expression of the phospho-deficient mutant of ECT2 at T341 suppressed the multinucleation induced by RNA interference to ECT2, indicating that ECT2 is biologically active even when it is not phosphorylated at T341. However, the phospho-mimic mutation at T341 weakly stimulates the catalytic activity of ECT2 as detected by serum response element reporter gene assays. As T341 is located at the hinge region of the N-terminal regulatory domain and C-terminal catalytic domain, phosphorylation of T341 may help accessing downstream signaling molecules to further activate ECT2. We found that the phospho-mimic mutantation T341D increases binding with itself or the N-terminal half of ECT2. These results suggest a conformational change of ECT2 upon phosphorylation at T341. This conformational change appears to relieve the auto-inhibition mechanism of ECT2. Therefore, ECT2 activity might be regulated by the phosphorylation status of T341. We propose that T341 phosphorylation by Cyclin B/Cdk1 could be a trigger for further activation of ECT2.Second, we showed that two mitotic kinases, Cdk1 and Plk1, phosphorylate ECT2 in vitro. We identified an in vitro Cdk1 phosphorylation site (T412) in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding was inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, did not exhibit strong association with Plk1 PBD compared with wild type ECT2. Moreover, ECT2 T412A, but not phospho-mimic T412D, displayed a diminished accumulation of GTP-bound RhoA compared with wild type ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of wild type ECT2 or the T412D mutant caused cortical hyperactivity in U2OS cells during cell division, this activity was not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert. Third, we showed that the inhibition of Cdk1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A, but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs.